Increased Rate of Yeast Cultivation from Packaged Beer with Environmentally Relevant Anaerobic Handling

ABSTRACT Beer production necessitates oxygen exclusion for the proper packaging and aging of the beer. Standard operating procedures, including those for quality testing, involve culturing microbes from packaged beer exposed to atmospheric oxygen, despite the generalized fact that packaged beer is an anaerobic environment. Our research goal was to apply an environmentally relevant culturing approach to improve yeast cultivation from bottled beer by attempting to ameliorate transplant shock. This is applicable to uniquely scrutinous quality assurance/control objectives and/or to grand cultivation goals, such as ancient beer samples. Although yeasts have the genetic capacity of oxygen protection, their epigenetic/biochemical states within anaerobic packaging may not adequately protect all cells from reactive oxygen species (ROS) at the moment of opening. Soon after opening, beer yeasts were found to be catalase negative, indicating deficient protection from at least one ROS. The general reduction/inhibition of growth was observed when the beer yeast was exposed to ROS in media, and atmospheric bottle opening was found to expose beer yeast to significantly increased levels of ROS. Our primary finding is that different oxygen handling methodologies (aerobic/microaerophilic/anaerobic) significantly impact the viable Saccharomyces yeast recovery rates of Bamberger’s Mahr’s Bräu Unfiltered Lager. Immediate anaerobic handling improved cultivation success rates, with significantly higher colony forming units (CFU)/mL being cultured, and reduced the volume of beer required to recover viable yeast. Aerobic standard operating procedures have mainly been developed to harvest yeast on large volumetric samples and/or samples with high viable cell numbers, but these procedures may be suboptimal and may underrepresent potential viable cell numbers. IMPORTANCE Procedures of beer production and packaging exclude oxygen to create a shelf-stable anaerobic environment, within which any viable organisms are stored. However, standard methodologies to cultivate microbes from such environments generally include opening in an oxygenated atmosphere. This study applies environmentally relevant culturing methods and compares the yeast recovery rates of beers handled in various oxygen conditions. When beer bottles were opened in anoxic conditions, higher colony counts were obtained, so a smaller volume of beer was required to recover viable cells. The yeast in beer, stored anaerobically, may not be biochemically prepared to fully protect cells from oxygen at the moment of opening. Negative catalase activity showed beer yeasts’ vulnerabilities to reactive oxygen. Atmospheric opening may reduce viability, causing the underreporting of viable cells. Anaerobic opening could increase the odds of successfully detecting/cultivating viable cell(s) that are present, which is pertinent to uniquely stringent quality screens and ambitious culturing attempts from rare samples.


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Increased rate of yeast cultivation from packaged beer with environmentally relevant anaerobic handling
The manuscript entitled "Increased rate of yeast cultivation from packaged beer with environmentally relevant anaerobic handling" suggests the anaerobic handling of beer bottles, as well as during the incubation of culture plates, to obtain a high number of CFU/mL. The authors suggested that high levels of ROS during aerobic manipulation negatively impact yeast development. The authors' hypothesis is confirmed by relatively simple yet efficient tests capable of determining that yeast recovery is affected when comparing the volumes of beer needed to obtain 30-300 colonies (an adequate number of colonies to CFU count) between protocols based on anaerobic or aerobic conditions. The manuscript was well written, clear, within the journal scope, and addresses interesting points of application of the technique. The methodology may be applied by several industries (mainly breweries) in QA/QC routine, as well as in the prospection of new isolates and/or recovery of yeasts from "historical" sources.
However, figures need attention, requiring important alterations. Some results need to be further explained, and current references (> 2018) should be added to the text.  Since Y axis has a considerably distance between CFU/mL numbers, it seems "Oxy/Oxy" and "Oxy/Micro" generated no yeast colonies even using ~80-130 mL of beer. The same was observed for "Ana/Micro" and "Ana/Ana" in (C), for which is impossible to determine how many mLs of beer were needed to 30-300 colonies.
Separating the Y axis into segments can help.
12. Figure  Increased rate of yeast cultivation from packaged beer with environmentally relevant anaerobic handling The manuscript entitled "Increased rate of yeast cultivation from packaged beer with environmentally relevant anaerobic handling" suggests the anaerobic handling of beer bottles, as well as during the incubation of culture plates, to obtain a high number of CFU/mL. The authors suggested that high levels of ROS during aerobic manipulation negatively impact yeast development. The authors' hypothesis is confirmed by relatively simple yet efficient tests capable of determining that yeast recovery is affected when comparing the volumes of beer needed to obtain 30-300 colonies (an adequate number of colonies to CFU count) between protocols based on anaerobic or aerobic conditions. The manuscript was well written, clear, within the journal scope, and addresses interesting points of application of the technique. The methodology may be Introduction 1. Lines 57-58: As for the presented value of the beer trade, I suggest adding updated data; 2021 data has been derived directly from the United Nations Comtrade Database and an up-todate estimate has been included (line 58).
2. Lines 65-66: Why is the ecosystem present in a beer bottle considered a relatively unstable community? Since it is usually composed of only one microorganism of only one species. The original verbiage oversimplified the ecological theory inversely relating species richness with ecosystem stability based on reduced functional redundancy. The text has been modified to be more elaborative and hopefully more accessible to more readers (line 64).
"Ecological theory inversely relates the number of species and their functional redundancies to ecological stability (8). A monocultured/single-species beer would probably be significantly impacted by environmental shifts due to being unlikely to be resilient on account of minimal functional redundancy." Results 3. Lines 118-119; 128-130: Could the small colonies identified on the anaerobically incubated plates be petite mutants? Could this influence the reuse of these yeasts in future productions using these yeasts?
Petite mutants are a genetic variant that yield small colony size essentially due to the genetic inability to grow aerobically even when oxygen is present (they can only grow anaerobically due to loss of function of the mitochondrial and/or host genes for oxidative phosphorylation). We forced yeasts to grow anaerobically thus shifting them into a similar state as petit mutants. But our small colony yeasts are not stable mutants that have lost the function to use oxygen, they've been deprived of oxygen. This reviewer's comment is a very valuable que to elaborate more about epigenetic shifts versus genetic mutations. The small colony sizes of our anaerobic incubated yeasts are an epigenetic phenotype, not related to a genetic mutation. This is showcased to be epigenetic when the colony size increases when a previously anaerobically incubated plate is later exposed to oxygen. If the yeasts were genetic mutants unable to oxidatively phosphorylate, then they would stay their small size even when increased oxygen is introduced. Elaborative comments have been added to the manuscript (lines 237-239, & 350) to orient the appreciation of epigenetics versus mutations. "This environmentally triggered increase in colony size suggests that the small colony size of our anaerobically incubated yeast is an epigenetic phenotypic variation related to current anaerobic metabolism, rather than a stable genetic mutation preventing oxygen utilization." "To test the cultivated yeasts' further tolerance and potential utilization of oxygen, plates with colonies were transferred to increased oxygen conditions." 4. Line 123: How much is "0,3 CFU/mL"? I suggest also presenting the standard deviation values; 0.3 CFU/ml, relates to the mathematical calculation that 80-120 ml of beer produced 30-300 colonies. One ml of beer would not produce even one colony, on average, so beer volumes tested needed to be increased and mathematically manipulated to the standard unit of per ml. The text has been edited to be more elaborative. "Countable plates were achieved for all conditions, average colony counts were quantified, and average CFU/ml were calculated based on the beer volume utilized" (line 119) Standard deviation values are warranted and have been added to the text (lines 122-126, 217-219).
5. Line 167: If possible, please add the CFU/mL concentration of the sample from which the "8 μL" was taken; A CFU/ml was not enumerated for the spot test technique because the sample was not spread across the 100m plate, as one would for a traditional spread plate for CFU enumeration. For this assay, inocula are deposited on a spot, rather than the whole plate. Standard dot-blot analysis of spot tests was performed for each growth spot, and data enumerated with the standard integrated density rather than CFU/ml. Each spot's integrated density was related to that of the control (YEPD standard medium). We tried to explain the general procedure of spot-tests that are commonly done: "In general, spot-tests compare the growth capabilities of organisms grown on different agar media by depositing a uniform volume of inoculum and observing the diameter and

opacity (integrated density) of the growth spot at the single location of inoculation" (line 400) "Conventional spot-tests were performed by depositing a uniform volume of inoculum and observing the diameter and opacity (integrated density) of the growth spot on agar after incubation." (line 167)
Materials and Methods 6. Line 301: How were the bottles stored before they were obtained? They come from unpasteurized beers, correct? Please clarify it in material and methods section; Yes, beers were unpasteurized and unfiltered, as a source of viable yeast. This has been modified to be more elaborative (line 374-309, 358 10. Scientific names must be in italics (e.g. Saccharomyces cerevisiae in some references); Indeed. The reference section's text has been modified accordingly. Since Y axis has a considerably distance between CFU/mL numbers, it seems "Oxy/Oxy" and "Oxy/Micro" generated no yeast colonies even using ~80-130 mL of beer. The same was observed for "Ana/Micro" and "Ana/Ana" in (C), for which is impossible to determine how many mLs of beer were needed to 30-300 colonies. Separating the Y axis into segments can help. The figure and figure caption have been modified. The axes have been 'broken' as the author suggests. One can see the data isn't at the zero mark, but it is still quite a dramatic difference in values. The figure caption now includes numerical data for more precise numerical data reference.
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